The organization of the genome into chromatin plays a central role in the regulation of developmental gene expression programs. One widely held belief to explain this regulation is the coordinated control of chromatin accessibility. However, there is little high resolution data measuring chromatin accessibility in vivo, the regulators of large scale chromatin accessibility are largely unknown, and the molecular mechanisms by which chromatin accessibility affects gene activity are unclear. This proposal describes a genomics approach to directly measure chromatin accessibility at high resolution in order to develop a system in the experimentally tractable Drosophila model that will examine the relationship between chromatin accessibility and gene activity. To accomplish this objective, we will utilize a technique developed by the Lieb lab termed FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements). We present data here not only demonstrating the ability of FAIRE to reproducibly isolate discrete regions of 'open'chromatin, but also to identify broad chromosomal regions that share chromatin accessibility properties, even using small numbers of cells. Specifically, we will first perform a genomics screen to identify novel regulators of chromatin accessibility by using RNAi-mediated knockdown in Drosophila S2 cells, followed by analysis of FAIRE-enriched DNA on microarrays. Second, we will generate genomic chromatin accessibility maps for different tissues and stages of Drosophila development by performing FAIRE followed by high-throughput sequencing, providing a framework to assess the relationship between chromatin accessibility and gene activity during development and identifying cis-regulatory modules that control developmental expression programs. Finally, we will begin to characterize potential regulators of chromatin accessibility in vivo through mutational analyses. Relevance to Public Health: Many developmental pathways and mechanisms, when misregulated, contribute to human disease. Therefore, the knowledge gained by these efforts will have applications in understanding mechanisms that underlie disease states such as cancer.